TCR analysis

We make use of scirpy to analyse scTCR-seq data.

Quality control

As a first step, we have a look at the "chain pairing" configuration, i.e how many CDR3 sequence have been detected per cell.

Here,

Multi-chains have already been removed in a previous filtering step.

Subset to cells with TCR only

UMAP before removal:

UMAP after removal:

Define clonotypes and clonotype clusters

For now, if a TCR has two alpha sequences, any of the two (plus the beta chain) needs to match an other cell to be considered a clonotype, or clonotype cluster, respectively

Analysis of clonotypes (as defined by nucleotide sequence identity)

Clonotype network (by identity)

The following plot visualizes the clonotypes. Each "blob" represents a clonotype, each dot a cell.

Clonotypes of size 1 ("singletons" or "non-expanded" clonotypes) are excluded for a better overview.

Diagnostics of cell-type annotation

public/private clonotypes

There are no public clonotypes:

clonal expansion by patient

The left panel shows the clonal expansion by the number of cells, the right panel by the number of clonotypes. I.e. if expansion is driven by a single, hyperexpanded clonotype, significant expansion will be visible in the left panel, but not in the right one.

Shannon diversity (normalized)

Analysis of clonotype clusters (by amino acid sequence alignment)

Notes and Limitations

Clonotype network

Public/Private clonotypes

Note that, based on nucleotide sequence identity, we did not find T cells with identical CDR3 sequences across patients. Therefore, the clonotypes identified as public here, consist of different, but highly similar CDR3 amino acid sequences.

contingency tables of cells

convergent clonotypes

We define a clonotype as convergent if multiple clonotypes with different nucleotide CDR3 sequences fall into the same "clonotype cluster", i.e. have similar amino acid sequences and likely recognize the same antigen.

This could be evidence of convergent evolution. See also scirpy glossary.

We observe convergence mostly among CD8+ T cells and among HPV- cells:

However, this seems mostly driven by Patient H143.

contingency tables and statistical tests (for CD8+ T cells)

Summary

Clonal expansion

We define "expanded" clonotypes as clonotypes with two (=2) or more than two (>= 3) cells.

Again, expanded clonotypes are mostly among CD8+ T cells. There are a few expanded regulatory T-cells as well.

Clonal expansion by cluster

Contingency tables and statistical tests (CD8+ T cells)

A simple differential gene expresion analysis of expanded vs. other CD8 T cells yields the following genes:

A simple differential gene expresion analysis of convergent vs. other CD8 T cells yields the following genes:

export clonotype table

Shannon entropy

Differential expression analysis of largest clonotype clusters

Cutoff 20

Cutoff 10

Cutoff 5

Differential gene expression analysis with cutoff 20

(for smaller cutoffs, the differential gene expression analysis does not work properly, as there will be to few cells per group)

Clonotype clusters by leiden cluster (cutoff 10)

For CD4 T cells only

Cutoff 10

Cutoff 3

Comparison of clonotype 4243 vs other Tregs

Plot marker genes

Store output